Cytotoxic boronic acid peptide analogs

ABSTRACT

A method is provided for inhibiting growth of cancer cells comprising contacting said cells with an effective growth-inhibiting amount of a compound of the formula (II): ##STR1## or a physiologically acceptable salt thereof, wherein A 1  and A 2  are individually L-amino acid residue selected from the group consisting of Ala, Pro, Gly, Glu, Leu, Lys, Phe, Ser, Vl, Ile, Arg, Tyr, Thr, Asp, Asn and Gly; R 1  is C 1  -C 6  (alkyl) which is unsubstituted or is substituted with an aromatic substituent or one or more in-chain bivalent groups selected from the group consisting of --O--, --CO--, --S--, --NH--, --CONH--, CH═CH--, and --SO 2  --; Y 1  and Y 2  are each H, or taken together form a moiety derived from a dihydroxy compound, and R 1  is H or an N-terminal protecting group.

This invention was made with government support under grant CA 09441awarded by the National Cancer Institute. The Government of the UnitedStates has certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATION

This is a division of application Ser. No. 07/574,294, filed Aug. 28,1990, now U.S. Pat. No. 5,106,948, which is a continuation-in-part ofU.S. application Ser. No. 07/199,891, filed May 27, 1988 (U.S. Pat. No.4,963,655).

FIELD OF THE INVENTION

This invention relates to boron analogs of amino acids and smallpeptides and the use of the analogs to inhibit growth or colonyformation of mammalian cells. More specifically, the present inventionrelates to methods to prepare boronic acid analogs of tripeptides ordipeptides, and their use to inhibit tumor growth in vivo and in vitro.

BACKGROUND OF THE INVENTION

Three major families of antitumor agents are known. Each of the familiesof agents is associated with a recognized mechanism of action. First,antitumor agents may be alkylating agents, which generally bind in acovalent manner with DNA to form bifunctional lesions. The bifunctionallesions involve adjacent or nearby bases on the same strand, oralternatively, involve bases on opposite strands forming interstrandcrosslinks. Second, antitumor agents may be antimetabolites, whichgenerally inhibit enzymes involved in the synthesis or assembly of DNA.Alternatively, an antimetabolite may serve as a fraudulent or analogsubstrate of DNA processes. Third, antitumor agents may be antibiotics,which work by intercalating into the DNA helix or introducing strandbreaks into DNA.

Thousands of potential anticancer agents have been evaluated.Essentially, all effective agents (of which very few have been found)appear to work by one of the above-mentioned mechanisms. The subjectinvention concerns a class of molecules which are not associated withany of the three major families of antitumor agents.

Proteases are ubiquitous enzymes involved in a myriad of cellularactivities including digestion, blood coagulation and fibrinolysis, theprocessing and degradation of proteins, sperm penetration, and have beenimplicated as important components in regulating cascades.

Proteases and protease inhibitors have been reported in association withcancer-related processes. The most common associations involve increasedprotease enzyme activities or enzyme concentration. Such increasedprotease activity may be associated with transformation of cells byviruses, chemicals or other agents, as well as with the metastaticpotential of cancer cells. Additionally, data have been published whichsuggest that protease inhibitors may prevent or reduce the incidence oftransformation and reduce the metastatic potential of cancer cells. Anumber of protease inhibitors has been previously evaluated againstmurine tumor cells in both culture and in whole animals as potentialantitumor agents. Reports exist of modest growth inhibition of cells inculture following exposure to protease inhibitors, such as chloromethylketones, soybean trypsin inhibitor, ovomucoid, and aprotinin. Most areinert or require very high concentrations to achieve significant tumorcell killing.

The synthesis and protease inhibition properties of a number ofdipeptide analogs, including Cbz-ala-1-borovaline andCbz-ala-1-borophenylalanine, wherein Cbz is an abbreviation for thebenzyloxycarbonyl protecting group, and Ala is the abbreviation for thealanyl peptidyl residue, have been previously reported (D. H. Kinder etal., J. Med. Chem., 28, 1917 (1985). The structures of these two analogsare depicted in FIG. 1. One of the analogs, Cbz-ala-1-borophenylalanine,has also been evaluated for its ability to inhibit cultured humannasopharyngeal carcinoma cells and Lewis lung murine tumor cells (Goz etal., Biochem. Pharmacol., 35, 3587 (1986)).

The synthetic route employed by D. H. Kinder et al. was based on theroute disclosed by D. Matteson et al. J. Amer. Chem. Soc., 103, 5241(1981) for the synthesis of (R)-1-acetamido-2-phenylethaneboronic acid.The synthetic route can be summarized as shown in Scheme 1, hereinbelow:##STR2## When, for example, R¹ is Cbz-ala, compound 1' isCbz-ala-1-borophenylalanine.

A. B. Shenvi et al. (U.S. Pat. No. 4,499,082) discloses tri- andtetrapeptide analogs of the general formula: ##STR3## wherein A¹, A² andA³ are amino acid residues, n is 1 and o is 0 or 1, Y¹ and Y² are eachH, or represent a divalent protecting group, R² is, e.g., alkyl oraralkyl and R¹ is H or an N-terminal protecting group such as Cbz.Specific tripeptide analogs are the subject of Examples 11, 16-19 and 22of this patent. These α-aminoboronic acid peptides are disclosed to beuseful as inhibitors of metallo, acid, and serine proteases. Suggestedtherapeutic uses for these compounds include the treatment of emphysema.A. B. Shenvi (U.S. Pat. No. 4,537,773) generally discloses and claimscompounds of formulas 2 and 3 as depicted in Scheme I, hereinabove,e.g., wherein the benzyl substituent has been replaced by lower alkyl.

However, a need exists for new synthetic routes to boronic acidpeptides. A further need exists for new therapeutic uses of boronic acidpeptides.

SUMMARY OF THE INVENTION

The present invention provides a therapeutic method to inhibit thereplication of cancer cells in vivo or in vitro by contacting saidcancer cells with an effective amount of certain α-aminoboronic acidpeptides. For example, the growth of cancer cells such as sarcoma,melanoma, leukemia or carcinoma cells can be inhibited in accord withthe present method. More specifically, the present invention provides amethod comprising the administration of an amount of certaindipeptidylaminoalkylboronic acids to a mammal, such as a human patient,afflicted with a cancer, in an amount effective to cure or amelioratesaid cancer, or the symptoms associated therewith. Thedipeptidylaminoalkylboronic acids, or "tripeptide analogs," which areuseful in the practice of the present invention, are of the generalformula (I): ##STR4## or a physiologically acceptable salt thereof,wherein x═y═1, A¹ and A² are individually L-amino acid residues selectedfrom the group consisting of Ala, Pro, Gly, Glu, Leu, Lys, Phe, Ser,Val, Ile, Arg, Tyr, Thr, Asp, Asn and Gly; R¹ is C₁ -C₆ (alkyl) which isunsubstituted or is substituted with an aromatic substituent or one ormore in-chain bivalent groups selected from the group consisting of--O--, --CO--, --S--, --NH--, --CONH--, --CH═CH-- and --SO₂ --; Y¹ andY² are each H, or taken together form a moiety derived from a dihydroxycompound having at least two hydroxy groups separated by at least twoconnecting atoms in a chain or ring, said chain or ring comprisingcarbon atoms, and optionally, a heteroatom or heteroatoms which can beN, S or O; with the proviso that when the heteroatom is O, R² cannot beH; and R² is H or an N-terminal protecting group. Preferably, Y¹ ═Y² ═H,R¹ is benzyl, methyl, isopropyl, 2-butyl, or isobutyl; most preferably,R¹ is benzyl or (C₁ -C₃)alkyl, A¹ is Ala, A² is Ala, Pro, Gly or Val andR² is an N-terminal protecting group such as Cbz or7-methoxycoumarin-4-ylacetyl.

The present invention also provides a general method for the synthesisof compounds of the formula I, and of dipeptide analogs of formula Iwherein x═o and y═1. The synthesis of dipeptide analogs of formula I inaccord with this route is depicted in FIG. 6. The synthesis of thetripeptide analog of formula 10 in accord with this route is depicted inFIG. 7. More generally, the present method comprises the steps of:

(a) converting a protected amino acid of the formula: R² --(A¹)--OH(15)into a compound of the formula R² --(A¹)--N₃ (18), wherein A¹ is asdefined hereinabove and R² is an N-terminal protecting group, so thatthe amido group (N₃ ) replaces the OH present on the CO₂ H group ofamino acid (A¹)OH;

(b) reacting the compound of the formula R² --(A¹)--N₃ (18) with (i) acompound of the formula: ##STR5## wherein A² and R¹ are as definedhereinabove and wherein Y¹ and Y² taken together form a moiety derivedfrom a dihydroxy compound as defined above to yield a compound of theformula (20): ##STR6## wherein Y¹, Y², R², A¹ , A² and R¹ are as definehereinabove for compounds 18 and 19; or, reacting compound (18) with(ii) a compound of the formula: ##STR7## wherein Y¹ and Y² are asdefined above for compound 9, and R₃, R₄, R₅, R₆, R₇ and R₈ areindividually (C₁ -C₄)alkyl, to yield a compound of formula: ##STR8##wherein R², A¹, R¹, Y¹ and Y² are as defined hereinabove for compounds18 and 19. Optionally, compounds 20 or 22 can be converted into thecorresponding boronic acids (Y¹ ═Y² ═H) by, e.g., cleavage of theboronate ester with BCl₃, and/or can be converted into the freepolypeptide (R² ═H) by removal of the N-terminal protecting group, e.g.,by hydrogenolysis.

Preferably, the amino acid acyl azide (18) is generated in a solventsystem including [bis(isopropyl)]-ethylamine (i-Pro₂ NEt), THF and DMF,by means of a reagent such as diphenylphosphoryl azide ((PhO)₂ P(O)N₃),followed by treatment with tetrabutylammonium fluoride-dihydrofluoride(n-Bu₄ NF.2HF).

As given herein, the abbreviations for the L-amino acid residues are inaccord with art-recognized nomenclature for peptidyl residues, as setforth in Shenvi et al., (U.S. Pat. No. 4,499,082) at Col. 4, line 53,through Col. 5, line 8, the disclosure of which is incorporated byreference herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the structure of the protease inhibitorsCbz-ala-1-borovaline and Cbz-ala-1-borophenylalanine. Synthesis andapparent K_(i) values are from Kinder and Katzenellenbogen, J. Med.Chem., 28, 1917 (1985).

FIG. 2 illustrates serine protease mechanism (upper panel) and inhibitormechanism (lower panel).

FIG. 3 illustrates colony Formation Assay. Human melanoma cells (A375)were exposed to Cbz-ala-1-borovaline or Cbz-ala-1-borophenylalanine for1 hour (FIG. 3A), 24 hours (FIG. 3B), or continuously (169 hours) (FIG.3C). Colonies were enumerated following 7 days of growth; each datapoint represents the average of 5 or more experiments.

FIG. 4 illustrates inhibition of protein synthesis in A375 cells inculture by Cbz-ala-1-borophenylalanine (0.075 μg/ml). Protein synthesiswas determined by the incorporation of [³ H]-leucine into protein inviable cells.

FIG. 5 illustrates inhibition of DNA synthesis in A375 cells in cultureby Cbz-ala-1-borophenylalanine (0 075 μg/ml). DNA synthesis wasdetermined by the incorporation of [³ H]-thymidine into viable cells.

FIG. 6 illustrates the synthetic Scheme I for production of a dipeptideanalog 1.

FIG. 7 illustrates the synthetic Scheme II for coupling of additionalamino acid residues or other moieties to an N-terminal end of thedipeptide analogs.

FIG. 8 illustrates boronic acid peptides which have been prepared.

DETAILED DESCRIPTION OF THE INVENTION

The synthetic method of the present invention is summarized below inScheme II: ##STR9## wherein A¹ and A² are individually L-amino acidresidues selected from the group consisting of Ala, Pro, Gly, Glu, Leu,Lys, Phe, Ser, Val, Ile, Arg, Tyr, Thr, Asp, Asn and Gly; R¹ is C₁₋₆(alkyl) which is unsubstituted or is substituted with an aromaticsubstituent or one or more in-chain bivalent groups selected from thegroup consisting of --O--, --CO--, --S--, --NH--, --CONH--, --CH═CH--and --SO₂ --; Y¹ and Y² taken together form a moiety derived from adihydroxy group having at least two hydroxy groups separated by at leasttwo connecting atoms in a chain or ring comprising carbon atoms, andoptionally, a heteroatom or heteroatoms which can be N, S or O; with theproviso that when the heteroatom is O, R² cannot be H; and R² is anN-terminal protecting group, and R₃, R₄, R₅, R₆, R₇, and R₈ areindividually C₁₋₄ (alkyl).

Therefore, compound 15 is an N-protected amino acid wherein thecarboxylic acid moiety is converted into an azidocarbonyl group byreaction with diphenylphosphoryl azide. Compound 18 is reacted in situwith 1-(bistrialkylsilyl)aminoboronic ester (21) in the presence oftetrabutyl ammonium fluoride-dihydrofluoride in DMF/THF to yield theN-protected boronic ester (22) which is referred to as a "dipeptideanalog." The synthesis of compound (18) is fully set forth in Shenvi etal. (U.S. Pat. No. 4,537,773), the disclosure of which is incorporatedby reference herein.

Alternatively, the N-protecting group of compound (22) can be removed,e.g., by hydrogenolysis, to yield a compound (19) which can be reactedwith compound 18 in the presence of THF/DMF to yield protectedtripeptide analog 20. The boronate ester can be cleaved in BCl₃ /CH₂ Cl₂to yield the boronic acid, and the N-terminal protecting group R² can beremoved by conventional methods. For example, Cbz groups can be removedby hydrogenolysis.

Therefore, compounds 20 or 22 prepared by the method of the inventionare peptide derivatives of α-aminoboronic acids, and are useful toinhibit the growth of mammalian cells, particularly cancer cells.

Each of the compounds of the invention comprise one or more, preferablyone to two, amino acids coupled to an acid-terminal α-aminoboronic acid,which can optionally be linked to a boron-terminal protecting group --Y¹--Y² --, as illustrated by the foregoing formula. The nature of theB-terminal protecting group --Y¹ --Y² -- can vary widely within thescope of the present invention. Suitable values for --Y¹ --Y² -- includemoieties derived from compounds, principally C₂ -C₁₂ diols, having atleast two hydroxy groups separated by at least two connecting atoms in achain or ring. Contemplated compounds within the foregoing descriptioninclude, for example, pinacol, perfluoropinacol, pinanediol, ethyleneglycol, diethylene glycol, catechol, 1,2-cyclohexanediol,1,3-propanediol, 2,3-butanediol, glycerol, diethanolamine and otheramino alcohols, and other equivalents apparent to those skilled in theart.

Preferably, the compounds of formula I that are useful in the presentinvention are free boronic acids (Y¹ ═Y² ═H), wherein R² is a N-terminalprotecting group. The phrase "N-terminal protecting group," as usedherein, refers to various animo-terminal protecting groups which can beemployed in peptide synthesis. Examples of suitable groups include acylprotecting groups, for example, formyl, dansyl, acetyl (Ac), benzoyl(Bz), trifluoroacetyl, succinyl (Suc) and methoxysuccinyl (MeOSuc);aromatic urethane protecting groups, for example, benzyloxycarbonyl(Cbz); and aliphatic urethane protecting groups, for example,tert-butoxycarbonyl (Boc) or adamantyloxycarbonyl. Gross and Mienhofer,eds., The Peptides, Vol. 3 (Academic Press, New York 1981), pp. 3-88,disclose numerous suitable amine protecting groups.

Compounds of the invention having side-chain amino groups, for example,where A¹, A² or A³ are Lys or Arg, can optionally comprise suitableN-terminal protecting groups attached to the side chains; similarly,amino acid residues having acidic or hydroxy side chains can beprotected in the form of benzyl or other suitable esters or ethers.

Pharmaceutically acceptable salts of the compounds of formula I, 20 or22 can be prepared by conventional methods useful to prepare aminesalts, and include the nontoxic salts of inorganic and organic acids,e.g., the hydrochloride, sulfate, methosulfate, tartrate, succinate,hydrobromide, and phosphate salts and the like.

One preferred synthetic pathway for the preparation of dipeptide analogsis shown in FIG. 6. The boroamino acid analogs 1 are preferably preparedin a stereospecific manner from (+)-α-pinanediol boronic esters 2. Thestereochemistry of the intermediate chloroboronic esters 3 and1-(bis-trimethylsilyl)aminoboronic esters 4 has been previouslydisclosed (see Matteson, J. Amer. Chem. Soc., 103, 5241 (1981)).

Utilizing the method of this invention, a wide variety of amino acidscan be coupled to pinanediol aminoboronic esters, thereby formingcompounds of the type 5 in yields of from about 40% to about 60%.

Cbz-protected amino acid acyl azides are generated in situ fromN-protected amino acids (R² OH) using diphenylphosphoryl azide in thepresence of a non-nucleophilic amine, preferably di-isopropyl ethylamine (i-Pro₂ NEt) followed by tetrabutyl ammoniumfluoride-dihydrofluoride (n-Bu₄ NF.2HF). The1-(bis-trimethylsilyl)aminoboronic esters 4 are added to Cbz-protectedamino acid acyl azides to remove the trimethylsilyl groups in dimethylformaldehyde/tetrahydrofuran (DMF/THF) solvent mixtures. Thenon-nucleophilic amine, i-Pro₂ NEt, is used as base in place oftriethylamine (Et₃ N) (a usual base in peptide coupling reactions) tominimize proteodeboronation facilitated by trimethylammonium salts asobserved in previous reactions. (See, for example, D. H. Kinder, J. Med.Chem., 28, 1917 (1985)).

The boronate esters, such as the pinanediol esters can be cleaved withabout 3 mole equivalents for BCl₃ in CH₂ Cl₂ at approximately 0° C. toyield the free boronic acids (1). The ester cleavage or hydrolysis is incontrast to previously published methods which rely upon destruction ofa pinanediol group to liberate the boronic acid. (See, for example, D.H. Kinder et al., cited above.) The hydrolysis reaction of thisinvention, however, is complete within about 5 minutes. The reaction issubsequently quenched by the addition of 1M NaOH which preventsreformation of the starting pinanediol ester from the liberatedpinanediol and aminoboronic acid.

The Cbz-deprotected dipeptide analogs can be prepared from 1 byhydrogenolysis in ethanol. The general reaction, illustrated by way ofexample for the compound ala-borovaline 6, is shown in FIG. 6. Theanalog 6 is produced in 76% yield after recrystallization from minimalvolumes of water.

Fluorescent Labeled and Tripeptide Analogs

7-Methoxycoumarin-4-ylacetyl analog 11, and tripeptide analog 10 can beprepared as shown for the borovaline analogs in FIG. 7. Followinghydrogenolysis of the Cbz group of 5 to yield 7, a suitably N-protectedamino acid (e.g., Cbz-alanine [8]) is coupled to the free amino group of7 using the acyl azide method described above to produce tripeptideanalog 10. Similarly, 7-methoxycoumarin-4-ylacetic acid (9) can becoupled to 7 to give the fluorescent dipeptide analog 11.

Removal of the pinanediol ester group can be accomplished as describedabove. However, in the particular case of the fluorescent analog 11,care must be taken to minimize destruction of the coumarin moiety byBCl₃. Specifically, the 7-methoxycoumarin-4-acetate group is convertedto 7-methoxy-4-methylcoumarin by BCl₃ in excess of the three equivalentsneeded for pinanediol ester cleavage or upon extended reaction times.

The dansyl analog of the dipeptide or tripeptide analogs can be preparedsimilarly by reaction of the deprotected amino group with5-dimethylamino-1-naphthalenesulfonyl chloride (dansylchloride).

Compounds That Have Been Prepared

FIG. 8 shows structures of molecules which have been prepared by themethod of the invention. An asterisk (*) denotes previous production byKinder et al., J. Med. Chem., cited above, by a different method.

It is believed that the method of the subject invention is also usefulin preparing additional compounds of the general type I. Specificallyenvisioned are compounds modeled on P₁ sites of substrates of elastaseand chymotrypsin, or other proteases frequently associated with tumorcells. Generally, the compounds are of the type R_(x) --R₁--NH--(P₁)CHB(OH)₂, where R_(x) is a hydrogen or an amino protectivegroup or fluorescent label, and R₁ and P₁ are amino acids or peptides,independently, in either the (d) or (1) configurations. The choice ofR_(I) is preferably based on known substrate specificity. The boronicamino acid analog is in the (R) configuration which corresponds to the(1)-amino acid. The (S) configuration (e.g., (d) amino acid analog) canbe established starting from (-)-α-pinanediol boronic esters asdescribed above.

For elastase, preferred P₁ side chains are: isopropyl(borovaline),2-butyl(boroisoleucine), 2-methylpropyl(boroleucine),methyl(boroalanine).

For α-chymotrypsin, preferred P₁ side chains are:benzyl(borophenylalanine), 4-hydroxybenzyl(borotyrosine), 3-CH₂-indole(borotyptophan).

For plasmin and plasminogen activator, preferred P₁ side chains are:2-guanidinoethyl(boroarginine), 4-aminopentyl(borolysine). For otherproteases, P₁ side chains include: 2-(methylthio)-ethyl(boromethionine),CH₂ COX (X═OH, boroaspartate, X═NH₂, boroasparagine) CH₂ CH₂ COX (X═OH,boroglutamate, X═NH₂, boroglutamine).

Therapeutic Applications

Methodologies and dosages useful to inhibit the growth of human cancercells in vitro are fully set forth in the Examples hereinbelow. Withrespect to in vivo cancer therapy, a number of vehicles and modes ofadministration are available to the art which can be employed toadminister one or more of the compounds of formula I to a mammal, suchas a human patient, afflicted with cancer.

For example, useful liquid vehicles which may be employed for theadministration of the present peptide anticancer drugs include theglycerol-ethanol solvent mixtures disclosed in U.S. patent applicationSer. No. 178,139 and the buffered aqueous glycerol solvents disclosed inCanadian Patent No. 1,252,717. Other useful liquid vehicles include theaqueous triglyceride emulsions disclosed by M. M. Ames et al., in CancerTreatment Reports, 66, 1579 (1982) and by R. L. Richardson in Proc.Amer. Assoc. Cancer Res., 27, 166 (1986). Of course, if a given boronicacid or ester peptide is sufficiently water-soluble, it may beadministered in aqueous solution, e.g., in a physiological salinesolution such as phosphate-buffered saline. See Lougheed et al.,Diabetologia, 19, 1 (1980).

The peptide solutions or dispersions can also be encapsulated inliposomes, and dispersions of liposomes in an appropriate liquid vehiclecan be parenterally administered, as disclosed by Suzuki et al. (U.S.Pat. No. 4,016,100), Jizomoto et al. (U.S. Pat. No. 4,673,567) and byRahman et al., Cancer Research, 42, 1817 (1982).

Parenteral or enteral administration of solutions or dispersions of thepresent peptide analogs can be employed, with intraperitoneal orintravenous injection or infusion being the preferred route. Forexample, a unit dosage of one or more of the present peptide analogs isdissolved or dispersed in the appropriate vehicle and infused into thebloodstream of the patient, e.g., over 0.5-18 hours, in order to achievedelivery of the desired amount of the drug in mg/kg of body weight.Although simple drip-type infusion can be employed, the use ofexternally or internally-placed infusion pumps permits substantiallycontinuous infusion of the peptide, and can maintain substantiallyconstant serum levels of the peptide. Useful infusion pumps for in vivodrug delivery are disclosed in Blackshear et al. (U.S. Pat. No.3,731,681), and in Dorman et al. (U.S. Pat. No. 4,772,263).

Total or unit dosages effective to inhibit the growth of the cells of aparticular cancer in vivo will be determined empirically, based upon theresponses observed in appropriate animal models and/or in clinicaltrials. Due to the high stability of the present analogs, as compared toother bioactive peptides, and their low toxicity, it is anticipated thatthe useful serum levels of the compounds can be varied widely, asdictated by the type and stage of the cancer or cancers to be treated.For example, unit doses may vary from about 0.1-0.2 mg/kg/day to as highas 50-250 mg/kg/day per day initially, with maintenance dosesadministered once or twice a week thereafter.

The invention will be further described by reference to the followingdetailed Examples.

Bioactivity

All of the molecules produced by the method of this invention areformally boronic acid analogs of dipeptides or tripeptides, although themethod can be readily adapted to prepared tetra-, penta- and higherpolypeptide analogs. One representative tripeptide analog of theinvention has been evaluated for its ability to inhibit the replicationof cancer cells. Also, a number of dipeptide analogs have been evaluatedas potential anticancer agents. It is believed that the analogs canfunction as protease inhibitors by mimicking the transition state ofserine protease-catalyzed peptide hydrolysis, as shown in FIG. 2.

Both the previously known and the new peptide analogs are inhibitors ofthe enzymes elastase and/or chymotrypsin (and quite possibly otherproteases). Additionally, the analogs are cytotoxic to human and murinetumor cells in culture. Thus, the analogs are expected to be useful asanticancer agents.

However, in vitro data on the analogs, most particularly the tripeptideanalog Cbz-ala-ala-boroalanine, (10a on FIG. 8) and the two dipeptideanalogs shown in FIG. 1, have been very striking by comparison withother inhibitors. Thus far, growth inhibition and colony formationinhibition of the two dipeptide analogs against a human melanoma cellline, a human lung carcinoma cell line, and a murine leukemia cell line(L1210) have been observed. The tripeptide analog 10a inhibits thegrowth of human A375 melanoma cells in vitro. In addition, the analogsdemonstrate a lack of inhibition of macromolecular synthesis,specifically DNA and protein synthesis.

Initial growth inhibition studies against L1210 murine leukemia cells inculture suggest that the boronic acid analogs of dipeptide compounds arevery cytotoxic. Following a 72-hour exposure to 0.03 uMala-1-borovaline, approximately 50% growth inhibition was observed. Thetwo compounds are also cytotoxic to human tumor cell lines. While notyet fully understood, the mechanism of cytotoxicity is not viamacromolecular synthesis inhibition in the human tumor cell lines.

The invention will be further described by reference to the followingdetailed examples. The numerals correspond to those on FIGS. 6 and 7.

EXAMPLE 1

Pinanediol Cbz-alanyl-borovaline (5, R¹ =iPr, R² =Cbz-Ala). Thebis-trimethylsilylaminoboronic ester (4) generated from the treatment ofthe 1-chloroboronic ester (31) with LiN(SiMe₃)₂ (0.1 mol) is reactedwith Cbz-Ala-N₃ generated from Cbz-Ala, diphenylphosphorylazide anddiisopropylethylamine at 0° C. in dimethylformamide (DMF), in a 1:1mixture of DMF:tetrahydrofuran with concomitant deprotection of theamino group with tetrabutylammonium fluoride-dihydrogenfluoride, for 18hrs (The ammonium fluoride salt provides an anhydrous source of fluorideion for deprotections. The material is also non-acidic, being preparedby the neutralization of n-Bu₄ NOH with 3 equivalents HF followed bydrying.) The reaction mixture is diluted with ether, and the organiclayer is consecutively washed with water, 1M HCl, 10% NaHCO₃, water, andbrine. The product is purified by flash chromatography on silica gel,using 50% ethyl acetate/hexane as eluting solvent, and is recrystallizedfrom hexane./ethyl acetate mixtures to yield 2.6 g of 5 (R¹ =iPr, R²=Cbz-Ala) (56%); NMR: δ (CDCl₃, 90 mHz) 7.3 (Ar, 5H, S), 6.5 (NH, 1H, d,J=7 Hz), 5.9 (NH, 1H, d, J=7 Hz), 5.0 (CH₂ Ar, 2H, 5) 4.2 (CHN, 1H, m),overlaps with 4.3 (CHO, 1H, d), pinanediol peaks at 2.5 (bm, 6H), 1.6,1.2, 0.9 (CH₃, 9H), 1.4 ((CH₃)₂ CH, 6H, d, J=8 Hz), 1.6 (CH₃ CHN, 3H, d,J=7 Hz).

EXAMPLE 2

Pinanediol Cbz-alanyl-alanyl-borovaline (10). The dipeptide 5, R¹ =iPr,R² =Cbz-Ala (250 mg) is treated with 50 mg 5% Pd/C under N₂ atmospherein 10 ml absolute ethanol. Hydrogen is then bubbled through the reactionmixture, and the reaction is monitored by TLC. Upon completion of thereaction, the flask is flushed with N₂, and catalyst is removed byfiltration through Celite. After evaporation of the solvent, thematerial is coupled to Cbz-Ala as described in Example 1, using theacylazide method. The reaction mixture is diluted with ether, and theorganic layer separated. The aqueous layer is washed with ethyl acetate,and the organic layers are combined. The organic layer is washed with10% bicarbonate, followed by 1M HCl, water and brine. After drying andevaporation of solvents, the product is recrystallized from ethylacetate./hexane mixtures. NMR:δ (CDCl₃) 7.3 (s, 5H), 6.7 (d, NH), 6.2(d, NH), 5.6 (d, B-CH-NH), 4.2 (m, CHN, --CHOB), 3.8 (dd, CH--B),(pinandiol peaks as Example 1), (1.6, d, CH₃ 6H) 1.4 (d, (CH₃)₂ CH, 6H).

EXAMPLE 3

Cbz-alanyl-alanyl-borovaline (10a). The protected tripeptide 10(typically 0.5-5 mmol) is added to a 0.3-0.5M solution (five-foldexcess) of BCl₃ in 1:2 CH₂ Cl₂ /dimethoxyethane (DME) prepared at -78°C., and allowed to stir for 2 hrs at 0° C. The disappearance of startingmaterial is followed using TLC. The reaction is quenched with water, andthe resulting mixture is diluted with ethyl acetate. The aqueous layeris separated and washed with ethyl acetate. The combined organic layersare washed with 1M NaOH (3×2 ml), and the basic extract is acidifiedwith 6M HCl. The product is extracted with ethyl acetate, dried, andsolvent evaporated. The product is recrystallized from acetone watermixtures after trituration of the oily residue with water to give a 33%yield of 10a as white crystals. NMR δ (DMSO-d₆) 7.2 (Ar, 5H, 5), 5.0(CH₂ Ar, 2H, 5), 4.1 (CHNH, 2H, m) 3.6 (CHB, 1H, d), 1.7 (CH(CH₃)₂, 1H,m), 1.5 ((CH₃ CH)₂, 6H, dd), 1.4 (CH₃)₂ CH, 6H, d, J=8 Hz), 2.5 (B(OH)₂,2.5H, bs).

EXAMPLE 4

Cbz-alanyl-prolyl-borovaline. (10c) was prepared in the same fashion as10a substituting Cbz-Pro for Cbz-Ala. The second coupling step is slowerbecause of the secondary proline nitrogen, and that step is usuallyallowed to proceed overnight. Physical characteristics were consistentwith the assigned structure.

EXAMPLE 5

Cbz-alanyl-alanyl-boroalanine. (10b) was prepared as for 10asubstituting the pinanediol 1-chloroethylboronate for the chloroboronicacid 3. Product gave physical characteristics consistent with theassigned structure.

EXAMPLE 6

Dansyl-alanyl-alanyl-borovaline. Cbz-alanyl-borovaline (10a) isdeprotected to the free amino compound by hydrogenolysis. 10a, 15 mg(0.04 mmol) is dissolved in ethanol, the system is flushed withnitrogen, and 2-5 mg 5% Pd/C catalyst is added. Hydrogen is bubbledthrough the solution for 45 min, after which the catalyst is removed byfiltration through Celite. The ethanol is removed under reducedpressure, and the residue is dried in vacuo overnight. The dried residueis dissolved in 1:1 acetone/-ethanol, and 11 mg dansyl chloride is addedwith 2-3 drops 10% NaHCO₃. The solution is allowed to stir overnight inthe dark. The solution is diluted with ether, washed with water, and theether is evaporated. The yellow solid is dissolved in acetone, andprecipitated with water, and after recovery of the solid, the process isrepeated two times. The yellow solid was isolated by filtration, driedunder vacuum, and gave 6 mg (35%) of product, mp 151°-152° C.; TLC, 30%methanol/ethyl acetate, R_(f) =0.8; NMR δ (CDCl₃, 90 mHz) 8.4-7.4 (Ar,6H, m), 6.7 (NH, 1H, d), 6.15 (NH, 1H, d), 5.65 (NH, 1H, d), 4.2 (CHN,2H, m), 3.75 (BCH, 1H, dd), 2.95 (N(CH₃)₂, 6H, s), 1.2 (CH₃ CHN, 6H,overlapping d), 0.95 ((CH₃)₂ CH, 6H, d), 1.9 ((CH₃)₂ CH, 1H, m), 1.7(B(OH)₂, 2H, bs).

EXAMPLE 7 Bioactivity A. Growth Inhibition Assay

Human melanoma cells (A375) were seeded at 2×10⁴ cells per tissueculture dish (60×15 mm, Falcon) in Delbecco's Minimal Essential Media(DMEM) containing 10% fetal calf serum and including 1% antibiotics.Cells were allowed to attach for 48 hours prior to exposure to drugs.Cbz-ala-1-borovaline and Cbz-ala-1-borophenylalanine were dissolved indimethyl sulfoxide (DMSO). The final DMSO concentration in media was0.5%. Fresh media was exchanged for the media including the drug atappropriate exposure times. Cells were counted after 24, 48 or 96 hoursof growth using a Coulter counter. Growth inhibition is expressed as thepercent reduction of drug treated cells with respect to untreatedcontrol cells.

B Colony Formation Assay (CFA)

Human melanoma cells (A375) were seeded at 500 cells per tissue culturedish and allowed to recover for 48 hours. The dipeptide or tripeptideand media mixture was prepared as described above usingCbz-ala-1-borovaline, Cbz-ala-1-borophenyl-alanine,7-0-methyl-coumarin-ala-1-boroval (11a),7-0-methylcoumarin-ala-1-borophe (11b) and Cbz-ala-ala-1-boroval (10a).Following peptide analog exposure, fresh media were substituted for thedrug and media mixture. After 7 days, colonies were stained with 0.25%Coomassie Blue and enumerated. Data are expressed as the percent ofcolonies present in treated plates with respect to control plates(percent survival).

C. Macromolecular Synthesis Studies

Human melanoma (A375) cells were seeded at 1×10⁵ cells per tissueculture dish 48 hours prior to the addition of the peptide analog (forexample, Cbz-ala-1-borovaline or Cbz-ala-1-borophenylalanine). The drugand media mixture were prepared as described above. During the last 30minutes of drug exposure, radioactive labeled precursors were added tothe media and drug mixture. Specifically, either ³ H-thymidine (0.2μCi/ml, to measure DNA synthesis), ³ H-uridine (0.2 μCi/ml, to measureRNA synthesis) or ³ H-leucine (1 μCi/ml, to measure protein synthesis)were added. Following 30 minutes at 37° C., the cells were harvested,resuspended in physiological buffered saline (PBS) and the cellscounted. An aliquot of the cell suspension was then lysed (10 mM Tris, 1mM EDTA, 0.1% SDS) and macromolecules were precipitated with ice-coldtrichloroacetic acid (TCA) (20%). Following three washes with TCA,radioactivity in the resulting pellet was counted on a Beckman LS2000Scintillation Counter. Data is expressed as the percentage of countsincorporated into 1×10⁶ treated cells as compared to 1×10⁶ untreatedcells (percent incorporation).

D. In Vivo Tumor Model

The highly invasive and metastatic B16-(BL6) murine melanoma wasselected as an in vivo model system. Experimental metastatic potentialof BL6 was determined after i.v. inoculation into the tail vein of B₆ D₂F₁ mice. Drugs which interfere with the metastatic process will impedethe colonization of the lungs by the tumors.

Tumors are grown in vitro to 90-95% confluence and are harvested withEGTA. Cells are injected i.v. into the tail vein of mice at aconcentration of 5×10⁴ viable cells in 0.2 ml Ca and Mg tris salinesolution. Mice are fed Purina rodent chow throughout the experiment.After three weeks, mice are sacrificed by cervical dislocation, lungsare removed en bloc, and preserved in Bouin's solution to facilitatevisualization of the tumors. At least 24 hrs following lung removal,lung tumor colonies are enumerated with the aid of a dissectionmicroscope. The number of tumors in the control groups and the treatmentgroups are then statistically compared using the Wilcoxin 2-sample test.

The drugs were formulated in 30% propylene glycol/sterile salinesolution for injection. Drugs were administered 24 hrs prior to tumorinjection and again 4-6 hrs post-tumor injection. Pretreatment ofanimals was designed to moderate any effect the protease inhibitorsmight have on NK cell activities. Control mice were injected with salinein the same volume as the treated mice.

E. Results 1. Inhibition of Growth/Colony Formation

As shown in Table 1, Cbz-ala-1-borovaline andCbz-ala-1-borophenylalanine were effective inhibitors of the growth andcolony formation of human melanoma (A375) cells in culture. Humanmelanoma (A375) cells were exposed to Cbz-ala-1-borovaline orCbz-ala-1-borophenylalanine for 1, 24 or 96 hours. Cells were countedfollowing 24, 48 or 96 hours of growth. Each data point represents theaverage of a minimum of 3 experiments. Colony formation data is fromFIG. 3C.

                                      TABLE 1                                     __________________________________________________________________________    Inhibition of A375 Growth and Colony Formation                                Exposure                                                                           Concen-                                                                             Growth Inhibition                                                                       Colony Formation                                                                        Apparent                                       Time tration                                                                             (%)       Inhibition                                                                         IC.sub.50                                                                          Ki                                             (Hours)                                                                            (μM)                                                                             24 hr                                                                            48 hr                                                                             96 hr                                                                            (%)  (μM)                                                                            (μM)                                        __________________________________________________________________________    Cbz-ala-1-Borovaline                                                           1   29.43 23 48  73 87                                                        1   7.36  14 33  46 41                                                       24   1.47  61 88  93 87                                                       24   .029  17 50  68 43                                                       24   .003   5  5  14  5   (0.91)                                                                             (0.77)(E).sup.1                                96   .736  53 78  89 --                                                       96   .029  21 51  68 --                                                       Cbz-ala-1-Borophenylalanine                                                    1   129   42 65  69 --                                                        1   51.6  20 41  52 55                                                       24   .645  75 72  98 97   (0.17)                                                                             (0.04)(C).sup.2                                24   .129  41 63  68 35                                                       24   .026   5  3  15 10                                                       96   .258  58 91  95 --                                                       96   .1239 38 68  75 --                                                       7-0 Methylcoumarin-ala-1-Borovaline (11a)                                     24    --   -- --  -- --   0.15 0.052(E).sup.                                  7-0-Methylcoumarin-ala-1-Borophenylalanine (11b)                              24   --    -- --  -- --   0.06 0.006(C).sup.                                  Cbz-ala-ala-1-Borovaline (10a)                                                24   --    -- --  -- --   0.05 0.018(C).sup.                                  Dansyl-ala-ala-Borovaline                                                     24   --    -- --  -- --   0.04 0.57 (C).sup.                                  __________________________________________________________________________     .sup.1 Elastase                                                               .sup.2 Chymotrypsin                                                      

Cbz-ala-1-borovaline was a more potent inhibitor of colony formationthan was Cbz-ala-1-borophenylalanine when assayed against human melanoma(A375) cells in culture (FIG. 3). Dose-dependent inhibition of colonyformation was observed for both analogs. Qualitatively similar resultswere obtained against human lung carcinoma (A549) cells in culture (datanot shown). A plateau was consistently observed with the 24-hourexposure to ala-1-borovaline between 0.029 μM and 0.833 μM (FIG. 3B).The plateau, however, was not observed in association with the analogafter the 1-hour (FIG. 3A) or continuous (FIG. 3C) exposure times. Asimilar plateau was never observed with Cbz-ala-1-borophenylalaninetreatment. A plateau was also seen with A549 cells.

Longer exposure times for both analogs led to enhanced inhibition ofcolony formation for all exposure times (FIG. 3), and enhancedinhibition of growth for the 1-hour versus 24-hour or 96-hour exposure(Table 1). The observed results are consistent with mechanisms involvingschedule dependent inhibition. Such mechanisms are possibly explained asa consequence of metabolism or processing of a drug to an activespecies. However, no difference was observed in the growth inhibitionfor 24-hour versus 96-hour exposure for either Cbz-ala-1-borovaline orCbz-ala-1-borophenylalanine (Table 1). The similarity of 24- and 96-hourexposure results on growth inhibition argues against schedule dependentinhibition.

The growth inhibition assay (Table 1) yielded qualitatively similarresults as the colony formation assay.

The growth inhibitory activity of other serine protease inhibitorsagainst human melanoma (A375) cells in culture was assessed (Table 2).

                  TABLE 2                                                         ______________________________________                                        Growth Inhibition Activity of                                                 Selected Protease Inhibitors                                                  Human melanoma (A375) cells were exposed to TPCK                              (N-tosyl-phenylalanine chloromethyl ketone),                                  ovomucoid or aprotinin for 96 hours. Fresh                                    inhibitor was added every 24 hours.                                           Data is from one representative experiment.                                   TPCK              Ovomucoid       Aprotinin                                   Conc.   %         Conc.   %       Conc. %                                     (μM) Inhib     (μM) Inhib   (μM)                                                                             Inhib                                 ______________________________________                                        284     100       89.3    5       15.4  0                                     142     100       17.9    0       1.54  0                                     28.4    100       3.57    0       .154  0                                     2.84     42       .357    1       .015  0                                     .284     9        .036    0                                                   .028     5        .004    0                                                   ______________________________________                                    

Negligible growth inhibition was observed following treatment with theinhibitors ovomucoid and aprotinin at concentrations at which the boronanalogs are potent inhibitors. Another serine protease inhibitor,N-tosylphenylalanine chloromethylketone (TPCK), inhibited growth at veryhigh concentrations. However, at lower inhibition (9% inhibition at0.284 μM) in the range, the boron analogs are very potent inhibitors(95% inhibition at 0.258 μM ala-1-borophenylalanine).

2. Inhibition of Synthesis of Macromolecules

Cbz-ala-1-borophenylalanine did not show an acute inhibition of proteinsynthesis at a concentration (0.075 μg/ml) which inhibited growth by 70%after 24-hour exposure (FIG. 4). This decrease in protein synthesisfollows cell survival and so most likely is a consequence of an injuredcell and not a primary mechanism leading to cell death.

3. In Vivo Tumor Inhibition

Tripeptide analog Cbz-ala-ala-boroval (10a) significantly reduced thenumber of tumors which colonized the lung following tumor injection, asshown on Table 3, below.

                  TABLE 3                                                         ______________________________________                                        Dose      Mean                                                                of 10a    # Tumors     Range   Significant                                    ______________________________________                                        Control   31.6         12-77   --                                             1.0 mg/kg 10.8          2-23   *                                              5.0 mg/kg 44.3         19-77   Toxicity, 6/10                                 ______________________________________                                         *Denotes stastically significant at p > 0.05. (Wilcoxon two sample test).

The dipeptide analog Cbz-ala-borophe also significantly reduced thenumber of tumors at 0.5, 1.0 and 2.0 mg/kg.

Preliminary tests of effective boron analogs administered to miceintravenously at doses of approximately 20 mg/kg did not revealsignificant toxicity or side effects.

EXAMPLE 8 Projected Phase I Studies of Boron Serine Protease InhibitorAnalogs A. Patients and Methodology

Protease inhibitor analogs 10a, 10b, and 10c are selected for clinicalevaluation based on broad antitumor activity against human tumor cellsin culture and in vivo activity against murine model tumors. The Phase Iclinical trial starting dose (22 mg/m²) is 1/10th the LD₁₀ dose in mice.Patients are treated by a 15-minute intravenous infusion once everythree weeks. Three new patients are evaluated at every dose. Patientswith tolerable toxicity who responded or had stable disease areretreated at the same dose until disease progression. Dose escalationsare determined by a modified Fibonacci scheme.

Twenty-four patients are enrolled in the three Phase I trials, threepatients at the first six dose levels, and six patients at the maximallytolerated (MTD) dose of 82 mg/m². All patients are ambulatory and in areasonable state of nutrition and have white blood cell counts greaterthan 4,000/mm², platelet counts greater than 130,000/m², hemoglobingreater than 10 g/dl, serum creatinine less than 1.5 mg/dl, serumalkaline phosphatase data 0, 1 or 2, SGOT levels less than 3 timesnormal and ECOG performance status less than 3. Patients are excluded ifthey present uncontrolled infection, persistent nausea and vomiting,chronic obstructive pulmonary disease, any neurological impairment,major surgery within the preceding 30 days, or radiation greater than15% of the bone marrow within 30 days. Patients are informed of theinvestigational nature of this study and an informed consent is requiredfor participation. Tests done prior to entry include a history, physicalexamination, tumor measurements when possible, WBC, hemoglobin, plateletcount, chest x-ray, electrocardiogram, electroencephalogram, urinalysis,SGOT and serum creatinine. Of the 24 patients, two have no priortreatment, 12 have had chemotherapy only, and 10 have had chemotherapyand radiotherapy. The most common malignancies are colorectal, lung,breast, and ovarian cancer. Patients with renal, melanoma, fibrosarcoma,and hepatic cancers are also treated in these studies.

Non-hematologic toxicities observed during the trials are Grade I-IInausea and vomiting, mucositis, and mild diarrhea. Myelosuppression isthe dose-limiting toxicity in this Phase I study. At higher doses,several patients exhibit platelet nadirs of approximately 50,000/mm² andwhite blood count nadirs 1,000-1,200/mm².

B. Compound 10a

Five of the twenty-four patients have objective evidence of antitumoractivity after treatment with boron protease inhibitor analog 10a. Twopatients with metastatic breast cancer with bone disease progressionfollowing previous therapy have objective evidence of tumor regression.They received five and eight cycles of treatment with the time totreatment failure of 7 months and 9+ months. Two patients with recurrentsmall cell lung cancer have objective tumor response. Time to treatmentfailure in these patients is four months and seven months. The lastresponding patient has metastatic melanoma with cytologically confirmedmetastases. The measurable breast metastases diminish in size over thenext six months until they are no longer measurable. Pulmonarymetastases are stable after 11+ months.

C. Compound 10b

Six of the twenty-four patients have objective evidence of antitumoractivity with the boron protease inhibitor analog 10b. Three patientswith metastatic breast cancer with bone disease progression followingprevious therapy have objective evidence of tumor regression. Theyreceive five and eight cycles of treatment with the time to treatmentfailure of 7 months, 8 months, and 9+ months. Two patients withrecurrent small cell lung cancer exhibit objective tumor response. Timeto treatment failure in these patients is four months and seven months.The last responding patient had metastatic melanoma with cytologicallyconfirmed metastases. The measurable breast metastases diminish in sizeover the next six months until they are no longer measurable. Pulmonarymetastases are stable after 11+ months.

D. Compound 10c.

Four of the twenty-four patients have objective evidence of antitumoractivity with the boron protease inhibitor analog. Two patients withmetastatic breast cancer with bone disease progression followingprevious therapy have objective evidence of tumor regression. Theyreceive five and eight cycles of treatment with the time to treatmentfailure of 7 months and 9+ months. One patient with recurrent small celllung cancer exhibits objective tumor response. Time to treatment failurein this patient is four months. The last responding patient hasmetastatic melanoma with cytologically confirmed metastases. Themeasurable breast metastases diminish in size over the next six monthsuntil they were no longer measurable. Pulmonary metastases are stableafter 11+ months.

These present compounds are also useful as antiviral agents, e.g.,against HIV, Herpes Simplex virus, and lentiviruses, as well asantiarthritic agents and antimalarial agents.

Although the present invention has been described with reference topreferred embodiments, workers skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention.

What is claimed is:
 1. A method for preparing a compound of formula (I):##STR10## wherein A¹ is an L-amino acid residue selected from the groupconsisting of Ala, Pro, Gly, Glu, Leu, Lys, Phe, Ser, Val, Ile, Arg,Tyr, Thr, Asp, Asn and Gly; R¹ is C₁₋₆ (alkyl) which is unsubstituted oris substituted with an aromatic substituent or one or more in-chainbivalent groups selected from the group consisting of --O--, --CO--,--S--, --NH--, --CONH--, --CH═CH-- and --SO₂ --; Y¹ and Y² takentogether form a moiety derived from a dihydroxy compound having at leasttwo hydroxy groups separated by at least two connecting atoms in a chainor ring comprising carbon atoms, and optionally, a heteroatom orheteroatoms which can be N, S or O; and R² is an N-terminal protectinggroup; comprising the steps of:(a) converting an N-protected amino acidof the formula R² --(A¹)--OH into a compound of the formula R² (A¹)--N₃wherein A¹ and R² are as defined above, and --OH represents the hydroxymoiety of the amino acid CO₂ H substituent; and (b) reacting thecompound of the formula R² (A¹)N₃ with a compound of the formula:##STR11## wherein Y¹, Y² and R¹ are as defined above, and R₃, R₄, R₅,R₆, R₇, and R₈ are individually C₁ -C₄ -(alkyl), under desilatingconditions to yield the compound of formula I.
 2. The method of claim 1further comprising cleaving said moiety derived from a dihydroxy groupto yield the compound of formula I wherein Y¹ ═Y² ═H.
 3. The method ofclaim 2 wherein said moiety is cleaved with BCl₃.
 4. The method of claim1 further comprising removing the N-terminal protecting group to yieldthe compound of formula I wherein R² ═H; with the proviso that theheteroatom or heteroatoms in the dihydroxy compound cannot be
 0. 5. Themethod of claim 2 further comprising removing the N-terminal protectinggroup to yield a compound of formula I wherein R² ═H.
 6. The method ofclaims 4 or 5 wherein said N-terminal protecting group is cleaved byhydrogenolysis.
 7. The method of claim 1 wherein R² (A¹)OH is reactedwith diphenylphosphonyl azide in the presence of diisopropylmethylaminein step (a).
 8. The method of claim 1 wherein step B is carried out inthe presence of tetrabutylammonium fluoride-dihydrofluoride.
 9. A methodof preparing a compound of formula (II): ##STR12## wherein A¹ and A² areindividually L-amino acid residues selected from the group consisting ofAla, Pro, Gly, Glu, Leu, Lys, Phe, Ser, Val, Ile, Arg, Tyr, Thr, Asp,Asn and Gly; R¹ is C₁₋₆ (alkyl) which is unsubstituted or is substitutedwith an aromatic substituent or one or more in-chain bivalent groupsselected from the group consisting of --O--, --CO--, --S--, --NH--,--CONH--, --CH═CH-- and --SO₂ --; Y¹ and Y² taken together form a moietyderived from a dihydroxy compound having at least two hydroxy groupsseparated by at least two connecting atoms in a chain or ring comprisingcarbon atoms, and optionally, a heteroatom or heteroatoms which can beN, S or O; and R² is an N-terminal protecting group; comprising thesteps of:(a) converting an N-protected amino acid of the formula R²(A¹)OH into a compound of the formula R² (A¹)N₃, wherein R² and A¹ areas defined above, and OH represents the hydroxy moiety of the amino acidCO₂ H substituent; and (b) reacting the compound of the formula R²(A¹)N₃ with a compound of the formula: ##STR13## wherein A², R¹, Y¹ andY² are as defined hereinabove, to yield the compound of formula II. 10.The method of claim 9 further comprising cleaving said moiety derivedfrom a dihydroxy group to yield a compound of the formula II, wherein Y¹═Y² ═H.
 11. The method of claim 10 wherein said moiety is cleaved withBCl₃.
 12. The method of claim 9 further comprising removing theN-terminal protecting group to yield the compound of formula II, whereinR² ═H; with the proviso that the heteroatom or heteroatoms in thedihydroxy compound cannot be
 0. 13. The method of claim 10 furthercomprising removing the N-terminal protecting group to yield a compoundof formula II wherein R² ═H.
 14. The method of claims 12 or 13 whereinsaid N-terminal protecting group is cleaved by hydrogenolysis.
 15. Themethod of claim 9 wherein R² (A¹)OH is reacted with diphenylphosphorylazide in the presence of diisopropylethylamine in step (a).